tides were 75% (E. gunnii), 76% (alfalfa), 67% (P. abies), 78%. (poplar), 76% (tobacco) ... Open reading frame of 1095 bp interrupted by four introns. The protein ...
Plant Physiol. (1995) 107: 285-286
Plant Gene Register
Genomic Nucleotide Sequence of an Arabidopsis fhaliana Gene Encoding a Cinnamyl Alcohol Dehydrogenase’ Marie Baucher, Jan Van Doorsselaere2, Jan Cielen, Marc Van Montagu*, Dirk Inzé, and Wout Boerjan Laboratorium voor Cenetica (M.B., J.V.D., J.G., M.V.M., W.B.), and Laboratoire Associé de I’lnstitut National de Ia Recherche Agronomique (France) (DA.), Universiteit Gent, B-9000 Cent, Belgium CAD (EC 1.1.1.195) catalyzes the conversion of cinnamaldehydes to the corresponding alcohols, the final step in the biosynthesis of the lignin precursors. CAD is considered one of the target enzymes for the modification of lignin biosynthesis (Whetten and Sederoff, 1991). This enzyme has been purified and characterized from severa1 plant species. cDNAs of CAD-encoding genes have been cloned from tobacco (Nicotiana tabacum) (Knight et al., 19921, Eucalyptus gunnii (Feuillet et al., 1993), Aralia cordata (Hibino et al., 1993), Pinus taeda (OMalley et al., 1992), Picea abies (Galliano et al., 1993), and poplar and alfalfa (Van Doorsselaere et al., 1995). Furthermore, a genomic clone has been obtained from Eucalyptus botryoides (Hibino et al., 1994). Here we report on the nucleotide sequence of the 3614-bp Arabidopsis tkaliana cad gene. The poplar cad cDNA (Van Doorsselaere et al., 1995) was used as a probe to screen a genomic library from A. tkaliana (Krebbers et al., 1988).A 5-kb HindIII fragment hybridizing with both the 5’ end and the 3‘ end of the poplar cad cDNA was isolated. This fragment was subcloned in pUC18 and sequenced. The analysis of the genomic clone revealed four introns and five exons encoding a polypeptide of 365 amino acids (Table I). The 5‘ sequenced region contains a putative TATA box and a CAT box at 126 bp and 200 b p upstream of the ATG, respectively. The cis element TAACGT, present in the anthocyanin biosynthesis genes in maize, could be identified. The calculated molecular mass of the encoded protein is 39.1 kD. The sequence identities with other CAD polypeptides were 75% ( E . gunnii), 76% (alfalfa), 67% (P. abies), 78% (poplar), 76% (tobacco), and 80% (A. cordata) at the amino acid level. Moreover, the A. tkaliana cad-coding sequence has 50% identity with the Eli3 gene from A. thaliana (Kiederowski et al., 1992). Eli3 also encodes an alcohol dehydrogenase and has a potential role in plant defense toward pathogens.
yable
Characteristics of the A. thaliana CAD gene
Organism: Arabidopsis thaliana ecotype C24. Cene Product; Pathway: CAD (EC 1.1.1.1 95), lignin biosynthesis, last step in monolignol synthesis. Techniques and Source: The genomic clone was isolated from a genomic library (Krebbers et al., 1988) using a full-length poplar cad cDNA (Van Doorsselaere et al., 1995; EMBL accession No. Z19568) as a probe. A 5-kb fragment (hybridizing with the 5’ and the 3’ ends of the poplar cad cDNA gene) was cloned into p U C l 8 for sequence analysis. Sequencing Strategy: Deletion subcloning (Nested Deletion Kit; Pharmacia, Uppsala, Sweden). Cene Identification: Sequence comparison of the deduced amino acid sequence with the full-length poplar cad cDNA and other cad sequences in the gene data bank. Clone Type: Cenomic sequence, full length (3614 bp), clone arabcad. Features of Gene Structure: Open reading frame of 1095 bp interrupted by four introns. Structural Features of the Protein: The protein consists of 365 amino acid residues. A consensus sequence for zinc-binding domain is found from Gly6’ to VaIa3. By comparison of the different CAD sequences mentioned in this paper, we could identify residues present in all alcohol dehydrogenases (Jornvallet al., 1987): three Gly (189, 201, and 204) important for coenzyme binding, two Cys (48 and 107) and one His (70) involved in Zn binding, and three amino acids with important binding properties (Asp”, Aspgo, and Glu7’). Conservation of the Ser (212 and 214) fits with the use of NADP+ as cofactor for CAD. The calculated molecular mass is 39.1 kD and the pl point is 5.32.
This supports the suggestion of Moershbacher et al. (1990) that CAD could also play a role in plant defense, its inhibition breaking resistance of wheat to stem rust.
This work was supported by grants from the Belgian Program on Interuniversity Poles of Attraction (Prime Minister’s Office, Science Policy Programming No. 38) and the Commission of European Communities (ECLAIR-OPLIGE No AGRE-0021-C [EDB]). Present address: Centre de Physiologie et Biologie Végétales, Université Paul Sabatier, 118 Route de Narbonne, F-31062 Toulouse Cedex, France. * Corresponding author; e-mail mamonQgengenp.rug.ac.be; fax 32-9-2645349.
ACKNOWLEDGMENTS
The authors wish to thank Nathalie Verbruggen and Thierry Wurch for critica1 reading of the manuscript, Luc Van Wiemeersch, Pierre Rouzé, and Jeroen Coppieters for help in the sequence analysis, and Martine De Cock for help with the manuscript. J.V.D. Abbreviation: CAD, cinnamyl alcohol dehydrogenase. 285
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is indebted to the Instituut ter Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw for a predoctoral fellowship. D.I. is a research Director of the Institut National de la Recherche Agronomique (France).
Received July 8, 1994; accepted July 15, 1994. Copyright Clearance Center: 0032-0889/95/107/0285/02. The EMBL accession number for the sequence reported in this article is 231715. LITERATURE CITED
Feuillet C, Boudet AM, Grima-Pettenati J (1993) Nucleotide sequence of a cDNA encoding cinnamyl alcohol dehydrogenase from Eucalyptus. Plant Physiol 103: 1447 Galliano H, Cabané M, Eckerskorn C, Lottspeich F, Sandermann H Jr, Ernst D (1993) Molecular cloning, sequence analysis and elicitor-/ozone-induced accumulation of cinnamyl alcohol dehydrogenase from Norway spruce (Picea abies L.). Plant Mo1Biol 2 3 145-156 Hibino T, Chen J-Q, Shibata D, Higushi T (1994) Nucleotide sequence of a Eucalyptus botryoides gene encoding cinnamyl alcohol dehydrogenase. Plant PhysiollO4: 305-306 Hibino T, Shibata D, Chen J-Q, Higuchi T (1993) Cinnamyl alcohol dehydrogenase from Aralia cordata: cloning of the cDNA and expression of the gene in lignified tissues. Plant Cell Physiol 3 4 659-665
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